Max von Gruber
Max von Gruber was an Austrian scientist. As a bacteriologist he discovered specific agglutination in 1896 with his English colleague Herbert Durham, but his main interests were studying sexual life. Max von Gruber was the son of Ignaz Gruber, a general practitioner and the first specialist in otology in Austria, publisher of a two-volume textbook on medical chemistry, his brother was Franz von Gruber. He graduated from the Schottengymnasium in Vienna and studied medicine at the University of Vienna, receiving his medical doctorate in 1876, he learned chemistry and physiology under Max von Pettenkofer and Karl von Voit in Munich and Karl Ludwig in Leipzig. Working under Pettenkofer was Hans Ernst August Buchner, who encouraged Gruber to concentrate on bacteriology. Unlike some of the great names of the time, among them Carl Wilhelm Nägeli, Theodor Billroth, Ferdinand Cohn, Robert Koch, Gruber recognized that bacteria possess a variability within limits determined by the culture medium; this theory was important for the differentiation of the categories of bacteria and gained significance for Gruber in his examinations of cholera vibrios, enabling him to distinguish them from other vibrios.
In 1882 Gruber was habilitated as a lecturer in Vienna, two years he became associate professor and head of the newly established Institute for Hygiene at the University of Graz. On 23 March 1887 he became ausserordentlicher professor in Vienna, succeeding Josef Nowak, on 10 December 1891 he was appointed to the chair of hygiene established in 1875 at the University of Vienna. Karl Landsteiner became his assistant in 1896. Another of his pupils, Alois Lode, in 1897 became the first professor in the new chair of hygiene at the University of Innsbruck; the working conditions in the Institute of Hygiene were so poor, that Gruber attempted to resign his chair and find employment as head of a laboratory in München or at the Jenner Institute in London, under Joseph Lister. It was while in Vienna, that Gruber, with his English student Herbert Edward Durham, discovered the agglutination which gained him international fame. Gruber left Vienna in 1902, in October that year he succeeded Hans Buchner as director of the Institute for Hygiene in München.
He held the post until his voluntary retirement on the occasion of his seventieth birthday. In Vienna he was succeeded by Arthur Schattenfroh, who held the chair from 1905 to 1923. During his last years, Gruber concentrated on his duties as president of the Bavarian Academy of Sciences. With Max Rubner and P. Martin Ficker he published the Handbuch der Hygiene. 6 volumes. As a leading racial hygienist, when he first met the Nazi dictator Adolf Hitler he described him as: It was the first time I had seen Hitler close at hand. Face and head of inferior type, cross-breed. Expression not of a man exercising authority in perfect self-command, but of raving excitement. At the end an expression of satisfied egotism. Über die als «Kommabacillen» bezeichneten Vibrionen von Koch und Finkler-Prior. Wiener medizinische Wochenschrift, 1885, 35, Nos 9–10: 261–264, 1907–301. Referring to Robert Koch, who established a nonsporulating, comma-shaped bacillus to be the causative agent of cholera. Dittmar Finkler and J.
Prior isolated Vibrio proteus from stools in a case of acute gastro-enteritis. Über active und passive Immunität gegen Cholera und Typhus, sowie über die bacteriologische Diagnose der Cholera und des Typhus. Wiener klinische Wochenschrift, 1896, 9: Nos 11–12: 183–186, 204–209. 14. Congress für Innere Medizin. Wiesbaden, 1896. Verhandlungen des Kongresses für innere Medizin, 1896: 207–227. Neue Früchte der Ehrlich’schen Toxinlehre. Wiener klinische Wochenschrift, 1903, 16: 791–793. Hygiene des Geschlechtslebens. Stuttgart, 1903. Wirkungsweise und Ursprung der aktiven Stoffe in den präventiven und antitoxischen Seris. Wiener klinische Wochenschrift, 1903, 16: 1097–1105. Schulärzte. Munich, 1905. Die Pflicht, gesund zu sein. Stuttgart, 1909. Fortpflanzung, Vererbung und Rassenhygiene. With Ernst Rüdin. Munich, 1911. Einleitung. Handbuch der Hygiene, volume 2, 1. Geschichte der Entdeckung der spezifischen Agglutination. In Rudolf Kraus and Constantin Levaditi, editors: Handbuch der Technik und Methodik der Immunitätsforschung.
Jena, 1914, I: 150–154. Lord Lister und Deutschland. Münchener medizinische Wochenschrift, 1927, 74: 592–593. Dankrede anlässlich der Feier seines 70. Geburtstages. Münchener medizinische Wochenschrift, 123: 70: 1038–1039. Hygiene of the sexual life Detailed Biography
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins known as antibodies, reacting with the proteins to be separated or characterized; the methods were used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis, crossed immunoelectrophoresis, rocket-immunoelectrophoresis, fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis ad modum Bøg-Hansen. Agarose as 1% gel slabs of about 1 mm thickness buffered at high pH is traditionally preferred for the electrophoresis as well as the reaction with antibodies; the agarose was chosen as the gel matrix because it has large pores allowing free passage and separation of proteins, but provides an anchor for the immunoprecipitates of protein and specific antibodies.
The high pH was chosen because antibodies are immobile at high pH. An electrophoresis equipment with a horizontal cooling plate was recommended for the electrophoresis. Immunoprecipitates may be seen in the wet agarose gel, but are stained with protein stains like Coomassie Brilliant Blue in the dried gel. In contrast to SDS-gel electrophoresis, the electrophoresis in agarose allows native conditions, preserving the native structure and activities of the proteins under investigation, therefore immunoelectrophoresis allows characterization of enzyme activities and ligand binding etc. in addition to electrophoretic separation. The immunoelectrophoretic analysis ad modum Grabar is the classical method of immunoelectrophoresis. Proteins are separated by electrophoresis antibodies are applied in a trough next to the separated proteins and immunoprecipitates are formed after a period of diffusion of the separated proteins and antibodies against each other; the introduction of the immunoelectrophoretic analysis gave a great boost to protein chemistry, some of the first results were the resolution of proteins in biological fluids and biological extracts.
Among the important observations made were the great number of different proteins in serum, the existence of several immunoglobulin classes and their electrophoretic heterogeneity. Crossed immunoelectrophoresis is called two-dimensional quantitative immunoelectrophoresis ad modum Clarke and Freeman or ad modum Laurell. In this method the proteins are first separated during the first dimension electrophoresis instead of the diffusion towards the antibodies, the proteins are electrophoresed into an antibody-containing gel in the second dimension. Immunoprecipitation will take place during the second dimension electrophorsis and the immunoprecipitates have a characteristic bell-shape, each precipitate representing one antigen, the position of the precipitate being dependent on the amount of protein as well as the amount of specific antibody in the gel, so relative quantification can be performed; the sensitivity and resolving power of crossed immunoelectrophoresis is than that of the classical immunoelectrophoretic analysis and there are multiple variations of the technique useful for various purposes.
Crossed immunoelectrophoresis has been used for studies of proteins in biological fluids human serum, biological extracts. Rocket immunoelectrophoresis is one-dimensional quantitative immunoelectrophoresis; the method has been used for quantitation of human serum proteins before automated methods became available. Fused rocket immunoelectrophoresis is a modification of one-dimensional quantitative immunoelectrophorsis used for detailed measurement of proteins in fractions from protein separation experiments. Affinity immunoelectrophoresis is based on changes in the electrophoretic pattern of proteins through specific interaction or complex formation with other macromolecules or ligands. Affinity immunoelectrophoresis has been used for estimation of binding constants, as for instance with lectins or for characterization of proteins with specific features like glycan content or ligand binding; some variants of affinity immunoelectrophoresis are similar to affinity chromatography by use of immobilized ligands.
The open structure of the immunoprecipitate in the agarose gel will allow additional binding of radioactively labeled antibodies to reveal specific proteins. This variation has been used for identification of allergens through reaction with IgE. Two factors determine that immunoelectrophoretic methods are not used. First they are rather require some manual expertise. Second they require rather large amounts of polyclonal antibodies. Today gel electrophoresis followed by electroblotting is the preferred method for protein characterization because its ease of operation, its high sensitivity, its low requirement for specific antibodies. In addition proteins are separated by gel electrophoresis on the basis of their apparent molecular weight, not accomplished by immunoelectrophoresis, but immunoelectrophoretic methods are still useful when non-reducing conditions are needed. Comprehensive text edited by Niels H. Axelsen in Scandinavian Journal of Immunology, 1975 Volume 4 Supplement Immunoelectrophoresis at the US National Library of Medicine Medical Subject Headings http://www.lib.mcg.edu/edu/esimmuno/ch4/immelec.htm Immuno-Electrophoresis.
A nephelometer is an instrument for measuring the concentration of suspended particulates in a liquid or gas colloid. A nephelometer measures suspended particulates by employing a light beam and a light detector set to one side of the source beam. Particle density is a function of the light reflected into the detector from the particles. To some extent, how much light reflects for a given density of particles is dependent upon properties of the particles such as their shape and reflectivity. Nephelometers are calibrated to a known particulate use environmental factors to compensate lighter or darker colored dusts accordingly. K-factor is determined by the user by running the nephelometer next to an air sampling pump and comparing results. There are a wide variety of research-grade nephelometers on the market as well as open source varieties; the main uses of nephelometers relate to air quality measurement for pollution monitoring, climate monitoring, visibility. Airborne particles are either biological contaminants, particulate contaminants, gaseous contaminants, or dust.
The chart to the left shows the sizes of various particulate contaminants. This information is helpful toward understanding the character of particulate pollution inside a building or in the ambient air, it is useful for understanding the cleanliness level in a controlled environment. Biological contaminants include mold, bacteria, animal dander, dust mites, human skin cells, cockroach parts, or anything alive or living at one time, they are the biggest enemy of indoor air quality specialists because they are contaminants that cause health problems. Levels of biological contamination depend on humidity and temperature that supports the livelihood of micro-organisms; the presence of pets, plants and insects will raise the level of biological contamination. Sheath air is clean filtered air that surrounds the aerosol stream to prevent particulates from circulating or depositing within the optic chamber. Sheath air prevents contamination caused by build-up and deposits, improves response time by containing the sample, improves maintenance by keeping the optic chamber clean.
The nephelometer creates the sheath air by passing air through a zero filter before beginning the sample. Nephelometers are used in global warming studies measuring the global radiation balance. Three wavelength nephelometers fitted with a backscatter shutter can determine the amount of solar radiation, reflected back into space through dust and particulate matter; this reflected light influences the amount of radiation reaching the earth's lower atmosphere and warming the planet. Nephelometers are used for measurement of visibility with simple one-wavelength nephelometers used throughout the world by many EPAs. Nephelometers, through the measurement of light scattering, can determine visibility in distance through the application of a conversion factor called Koschmieder's formula. In medicine, nephelometry is used to measure immune function. Gas-phase nephelometers are used in the detection of smoke and other particles of combustion. In such use, the apparatus is referred to as an aspirated smoke detector.
These have the capability to detect low particle concentrations and are therefore suitable to protecting sensitive or valuable electronic equipment, such as mainframe computers and telephone switches. Because optical properties depend on suspended particle size, a stable synthetic material called "Formazin" with uniform particle size is used as a standard for calibration and reproducibility; the unit is called Formazin Turbidity Unit. Nephelometric Turbidity Units specified by United States Environmental Protection Agency is a special case of FTU, where a white light source and certain geometrical properties of the measurement apparatus are specified. Formazin Nephelometric Units, prescribed for 9 measurements of turbidity in water treatment by ISO 7027, another special case of FTU with near infrared light and 90° scatter. Formazin Attenuation Units specified by ISO 7027 for water treatment standards for turbidity measurements at 0° a special case of FTU. Formazin Backscatter Units, not part of a standard, is the unit of optical backscatter detectors, measured at c.
180° a special case of FTU. European Brewery Convention turbidity units Concentration Units Optical Density Jackson "Candle" Turbidity Units Helms Units American Society of Brewing Chemists turbidity units Parts Per Million of standard substance, such as PPM/DE "Trübungseinheit/Formazin" a German standard, now replaced by the FNU unit. Diatomaceous earth an older standard, now obsoleteA more popular term for this instrument in water quality testing is a turbidimeter. However, there can be differences between models of turbidimeters, depending upon the arrangement of the source beam and the detector. A nephelometric turbidimeter always monitors light reflected off the particles and not attenuation due to cloudiness. In the United States environmental monitoring the turbidity standard unit is called Nephelometric Turbidity Units, while the international standard unit is called Formazin Nephelometric Unit; the most applicable unit is Formazin Turbidity Unit, although different measurement methods can give quite different values as reported in FTU.
Gas-phase nephelometers are used to study the atmosphere. These can provide information on atmospheric albedo. ISO 7027 Water purification
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, therefore allows visualization of the distribution of the target molecule through the sample; the specific region an antibody recognizes on an antigen is called an epitope. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary. Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a used example of immunostaining and is a specific example of immunohistochemistry; this technique makes use of fluorophores to visualise the location of the antibodies. Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, may be used to analyze the distribution of proteins and small biological and non-biological molecules.
This technique can be used to visualize structures such as intermediate-sized filaments. If the topology of a cell membrane has yet to be determined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures. Immunofluorescence can be used as a "semi-quantitative" method to gain insight into the levels and localization patterns of DNA methylation since it is a more time consuming method than true quantitative methods and there is some subjectivity in the analysis of the levels of methylation. Immunofluorescence can be used in combination with other, non-antibody methods of fluorescent staining, for example, use of DAPI to label DNA. Several microscope designs can be used for analysis of immunofluorescence samples. Various super-resolution microscope designs that are capable of much higher resolution can be used. To make fluorochrome-labeled antibodies, a fluorochrome must be conjugated to the antibody. An antigen can be conjugated to the antibody with a fluorescent probe in a technique called fluorescent antigen technique.
Staining procedures can apply to both fixed antigen in the cytoplasm or to cell surface antigens on living cells, called "membrane immunofluorescence". It is possible to label the complement of the antibody-antigen complex with a fluorescent probe. In addition to the element to which fluorescence probes are attached, there are two general classes of immunofluorescence techniques: primary and secondary; the following descriptions will focus on these classes in terms of conjugated antibodies. There are two classes of immunofluorescence techniques and secondary. Primary immunofluorescence uses a primary antibody, chemically linked to a fluorophore; the primary antibody recognizes the target molecule and binds to a specific region called the epitope. The attached fluorophore can be detected via fluorescent microscopy, depending on the messenger used, will emit a specific wavelength of light once excited. Direct immunofluorescence, although somewhat less common, has notable advantages over the secondary procedure.
The direct attachment of the messenger to the antibody reduces the number of steps in the procedure, saving time and reducing non-specific background signal. This limits the possibility of antibody cross-reactivity and possible mistakes throughout the process. However, some disadvantages do exist in this method. Since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence and may result in false negatives. Direct immunofluorescence requires the use of much more primary antibody, expensive, sometimes running up to $400.00/mL. Secondary immunofluorescence uses two antibodies. Multiple secondary antibodies can bind a single primary antibody; this provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the primary protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.
This protocol is possible because an antibody consists of two parts, a variable region and constant region. It is important to realize that this division is artificial and in reality the antibody molecule is four polypeptide chains: two heavy chains and two light chains. A researcher can generate several primary antibodies that recognize various antigens, but all share the same constant region. All these antibodies may therefore be recognized by a single secondary antibody; this saves the cost of modifying the primary antibodies to directly carry a fluorophore. Different primary antibodies with different constant regions are generated by raising the antibody in different species. For example, a researcher might create primary antibodies in a goat that recognize several antigens, employ dye-coupled rabbit secondary antibodies that recognize
Epitope mapping is the process of experimentally identifying the binding site, or "epitope", of an antibody on its target antigen. Identification and characterization of antibody binding sites aid in the discovery and development of new therapeutics and diagnostics. Epitope characterization can help elucidate the mechanism of binding for an antibody and can strength intellectual property protection. Experimental epitope mapping data can be incorporated into robust algorithms to facilitate in silico prediction of B-cell epitopes based on sequence and/or structural data. Epitopes are divided into two classes: linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but brought together upon three-dimensional protein folding. B-cell epitope mapping studies suggest that most interactions between antigens and antibodies autoantibodies and protective antibodies, rely on binding to conformational epitopes.
By providing information on mechanism of action, epitope mapping is a critical component in therapeutic monoclonal antibody development. Epitope mapping can reveal how a mAb exerts its functional effects - for instance, by blocking the binding of a ligand or by trapping a protein in a non-functional state. Many therapeutic mAbs target conformational epitopes that are only present when the protein is in its native state, which can make epitope mapping challenging. Epitope mapping has been crucial to the development of vaccines against prevalent or deadly viral pathogens, such as chikungunya, dengue and Zika viruses, by determining the antigenic elements that confer long-lasting immunization effects. Complex target antigens, such as membrane proteins and multi-subunit proteins are key targets of drug discovery. Mapping epitopes on these targets can be challenging because of the difficulty in expressing and purifying these complex proteins. Membrane proteins have short antigenic regions that fold only when in the context of a lipid bilayer.
As a result, mAb epitopes on these membrane proteins are conformational and, are more difficult to map. Epitope mapping has become prevalent in protecting the intellectual property of therapeutic mAbs. Knowledge of the specific binding sites of antibodies strengthens patents and regulatory submissions by distinguishing between current and prior art antibodies; the ability to differentiate between antibodies is important when patenting antibodies against well-validated therapeutic targets that can be drugged by multiple competing antibodies. In addition to verifying antibody patentability, epitope mapping data have been used to support broad antibody claims submitted to the United States Patent and Trademark Office. Epitope data have been central to several high-profile legal cases involving disputes over the specific protein regions targeted by therapeutic antibodies. In this regard, the Amgen v. Sanofi/Regeneron PCSK9 inhibitor case hinged on the ability to show that both the Amgen and Sanofi/Regeneron therapeutic antibodies bound to overlapping amino acids on the surface of PCSK9.
There are several methods available for mapping antibody epitopes on target antigens: X-ray co-crystallography and cryogenic electron microscopy. X-ray co-crystallography has been regarded as the gold-standard approach for epitope mapping because it allows direct visualization of the interaction between the antigen and antibody. Cryo-EM can provide high-resolution maps of antibody-antigen interactions. However, both approaches are technically challenging, time-consuming, expensive, not all proteins are amenable to crystallization. Moreover, these techniques are not always feasible due to the difficulty in obtaining sufficient quantities of folded and processed protein. Neither technique can distinguish key epitope residues for mAbs that bind to the same group of amino acids. Array-based oligo-peptide scanning. Known as overlapping peptide scan or pepscan analysis, this technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein, tests for their ability to bind the antibody of interest.
This method is fast inexpensive, suited to profile epitopes for large numbers of candidate antibodies against a defined target. The epitope mapping resolution depends on the number of overlapping peptides; the main disadvantage of this approach is that it cannot be used to obtain conformational epitopes, which are the most relevant epitope type for human therapeutic mAbs. However, one study mapped discontinuous epitopes on CD20 using array-based oligo-peptide scanning, by combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide. Site-directed mutagenesis mapping; the molecular biological technique of site-directed mutagenesis can be used to enable epitope mapping. In SDM, systematic mutations of amino acids are introduced into the sequence of the target protein. Binding of an antibody to each mutated protein is tested to identify the amino acids that comprise the epitope; this technique can be used to map both linear and conformational epitopes but is labor-intensive and time-consuming limiting analysis to a small number of amino-acid residues.
High-throughput shotgun mutagenesis epitope mapping. Shotgun mutagenesis is a
White blood cell
White blood cells are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Leukocytes are found throughout the body, including lymphatic system. All white blood cells have nuclei, which distinguishes them from the other blood cells, the anucleated red blood cells and platelets. Types of white blood cells can be classified in standard ways. Two pairs of broadest categories classify them either by cell lineage; these broadest categories can be further divided into the five main types: neutrophils, basophils and monocytes. These types are distinguished by their physical and functional characteristics. Monocytes and neutrophils are phagocytic. Further subtypes can be classified; the number of leukocytes in the blood is an indicator of disease, thus the white blood cell count is an important subset of the complete blood count.
The normal white cell count is between 4 × 109/L and 1.1 × 1010/L. In the US, this is expressed as 4,000 to 11,000 white blood cells per microliter of blood. White blood cells make up 1% of the total blood volume in a healthy adult, making them less numerous than the red blood cells at 40% to 45%. However, this 1 % of the blood makes a large difference to health. An increase in the number of leukocytes over the upper limits is called leukocytosis, it is normal. It is abnormal, when it is neoplastic or autoimmune in origin. A decrease below the lower limit is called leukopenia; this indicates a weakened immune system. The name "white blood cell" derives from the physical appearance of a blood sample after centrifugation. White cells are found in the buffy coat, a thin white layer of nucleated cells between the sedimented red blood cells and the blood plasma; the scientific term leukocyte directly reflects its description. It is derived from the Greek roots leuk- meaning "white" and cyt- meaning "cell".
The buffy coat may sometimes be green if there are large amounts of neutrophils in the sample, due to the heme-containing enzyme myeloperoxidase that they produce. All white blood cells are nucleated, which distinguishes them from the anucleated red blood cells and platelets. Types of leukocytes can be classified in standard ways. Two pairs of broadest categories classify them either by cell lineage; these broadest categories can be further divided into the five main types: neutrophils, basophils and monocytes. These types are distinguished by their physical and functional characteristics. Monocytes and neutrophils are phagocytic. Further subtypes can be classified. Granulocytes are distinguished from agranulocytes by their nucleus shape and by their cytoplasm granules; the other dichotomy is by lineage: Myeloid cells are distinguished from lymphoid cells by hematopoietic lineage. Lymphocytes can be further classified as T cells, B cells, natural killer cells. Neutrophils are the most abundant white blood cell, constituting 60-70% of the circulating leukocytes, including two functionally unequal subpopulations: neutrophil-killers and neutrophil-cagers.
They defend against fungal infection. They are first responders to microbial infection, they are referred to as polymorphonuclear leukocytes, although, in the technical sense, PMN refers to all granulocytes. They have a multi-lobed nucleus; this gives the neutrophils the appearance of having multiple nuclei, hence the name polymorphonuclear leukocyte. The cytoplasm may look transparent because of fine granules. Neutrophils are active in phagocytosing bacteria and are present in large amount in the pus of wounds; these cells are not able to die after having phagocytosed a few pathogens. Neutrophils are the most common cell type seen in the early stages of acute inflammation; the life span of a circulating human neutrophil is about 5.4 days. Eosinophils compose about 2-4% of the WBC total; this count fluctuates throughout the day and during menstruation. It rises in response to allergies, parasitic infections, collagen diseases, disease of the spleen and central nervous system, they are rare in the blood, but numerous in the mucous membranes of the respiratory and lower urinary tracts.
They deal with parasitic infections. Eosinophils are the predominant inflammatory cells in allergic reactions; the most important causes of eosinophilia include allergies such as asthma, hay fever, hives. They secrete chemicals that destroy these large parasites, such as hook worms and tapeworms, that are too big for any one WBC to phagocytize. In general, their nucleus is bi-lobed; the lobes are connected by a thin strand. The cytoplasm is full of granules that assume a characteristic pink-orange color with eosin stain
Direct fluorescent antibody
A direct fluorescent antibody known as "direct immunofluorescence", is an antibody, tagged in a direct fluorescent antibody test. Its name derives from the fact that it directly tests the presence of an antigen with the tagged antibody, unlike western blotting, which uses an indirect method of detection, where the primary antibody binds the target antigen, with a secondary antibody directed against the primary, a tag attached to the secondary antibody. Commercial DFA testing kits are available, which contain fluorescently labelled antibodies, designed to target unique antigens present in the bacteria or virus, but not present in mammals; this technique can be used to determine if a subject has a specific viral or bacterial infection. In the case of respiratory viruses, many of which have similar broad symptoms, detection can be carried out using nasal wash samples from the subject with the suspected infection. Although shedding cells in the respiratory tract can be obtained, it is in low numbers, so an alternative method can be adopted where compatible cell culture can be exposed to infected nasal wash samples, so if the virus is present it can be grown up to a larger quantity, which can give a clearer positive or negative reading.
As with all types of fluorescence microscopy, the correct absorption wavelength needs to be determined in order to excite the fluorophore tag attached to the antibody, detect the fluorescence given off, which indicates which cells are positive for the presence of the virus or bacteria being detected. Direct immunofluorescence can be used to detect deposits of immunoglobulins and complement proteins in biopsies of skin and other organs, their presence is indicative of an autoimmune disease. When skin not exposed to the sun is tested, a positive direct IF is an evidence of systemic lupus erythematosus. Direct fluorescent antibody can be used to detect parasitic infections, as was pioneered by Sadun, et al.. Immunofluorescence Direct+Fluorescent+Antibody+Technique at the US National Library of Medicine Medical Subject Headings